P-023 Physics-Driven Microfluidic Pathway for Enhanced Sperm Filtration

Study question Could the dysregulation of the inflammatory response in the aged ovary be associated with foamy cells generation and be ameliorated using an alternative therapeutic treatment? Summary answer Aged ovarian macrophages show an inflammatory profile associated with incorporation of lipid droplets that can be reduced after culture with platelet-rich plasma from fertile women. What is known already The concept of ‘inflammaging’ represents the inflammation associated to aging, which is systemic, chronic and linked to the increase of pro-inflammatory mediators. This microenvironment is associated with the presence of ovarian foamy macrophages, a unique subset with intracytoplasmic lipids accumulation. Here, we investigate the mechanisms underlying the exacerbated inflammatory response during ovarian aging, focusig on the generation of foamy macrophages and their impact in oocyte quality. Since, oocytes develop in the context of ovarian follicles, we obtained follicular fluid (FF) from women with different ages, to investigate ovarian immune microenvironment and their modulation after in vitro platelet-rich plasma (PRP) treatment. Study design, size, duration Mononuclear cells were recovered from FF from women of different ages and IL-1β production and macrophages and lymphocytes profiles tested by immune-staining and FACS analysis. To test foamy cells generation, we measured surface and intracellular CD68 expression (scavenger receptor involved in lipid accumulation). Immune cells profiles were correlated with clinic parameters as number of oocytes, M2 and atretic follicles. Finally, isolated ovarian macrophages were treated with PRP and modulation of macrophage profile was tested. Participants/materials, setting, methods We studied FF (n = 70) from women from ovodonation (21-29y), with cryopreservation desire (30-35y) and ART indication (36-42y). The San Isidro Ethics Committee, Buenos Aires, Argentina, approved this study. Mononuclear cells were isolated using Ficoll-Hypaque and macrophages profile (IL-1β+, CD68+) and the novel lymphocyte regulatory subpopulation (CD3+CD4-CD8-) tested by flow cytometry. Lipid droplets were measured using BODIPY 493/503 probe. Ovarian macrophages were isolated by adherence and treated with PRP (obtained from fertile women) for 5 days. Main results and the role of chance FF macrophages from aged women (35-42y) showed a significant increase in IL-1β production in comparison with the youngest group (21-29y), that also correlates negatively with the number of M2 oocytes recovered. Focusing on macrophage characterization, FF from aged women displayed an increased frequency of CD68+ macrophages in comparison with young women. Since CD68 shuttles between the cytoplasm and the plasmatic membrane to incorporate lipids, we evaluated lipid accumulation in FF macrophages to generate foamy cells. Interestingly, we found an increase of lipid droplets in FF macrophages from aged women in comparison with the younger group. Therefore, we discriminated surface and intracellular CD68 detection and found higher intracellular CD68 expression in aged macrophages, correlating with lipid droplets accumulation. Then, we isolated ovarian macrophages from aged women and treated with fertile PRP to evaluate its modulatory effects and observed a reduction in the frequency of CD68+IL-1β cells in comparison with those cultures without treatment. Finally, we focused on a novel T lymphocyte regulatory subpopulation, CD3+CD4-CD8- (DNT), that is emerging as effector cells capable of mediating immune tolerance in the female reproductive system. We found that FF from aged women displayed a reduced frequency of DNT, in comparison with FF from younger women. Limitations, reasons for caution The present results were obtained using FF samples from women from different ages. Even though the samples represent the ovarian microenvironment, it is difficult to study the human ovary per se and further studies are necessary to elucidate whether these mechanisms operate similarly in vivo. Wider implications of the findings We demonstrated that dysregulation of the inflammatory response during aging leads to the generation of foamy cells and reduction of DNT cells associated to the regulation of the immune ovarian microenvironment impacting in oocyte quality. In vitro PRP treatment displayed immunomodulatory effects, suggesting its relevance as a potential therapeutic treatment. Trial registration number not applicable

Follicular curetting is the gentle and rapidly rotating movement of the aspiration needle in a clockwise and counterclockwise fashion. The aim of our study is to assess this retrieval technique comparing cycle and pregnancy outcomes between curetting and non-curetting. This was a retrospective review of 817 patients that underwent oocyte retrieval at our fertility center from January 1st, 2016 until August 31st, 2019. All patients enrolled in the study underwent standard ovarian stimulation protocols. Three physicians routinely utilized follicle curetting during oocyte retrieval, while a fourth physician did not curette. Retrievals and embryo transfers were performed based on a rotating physician schedule. Retrievals that were performed using a curetting technique had a significantly higher number of total oocytes retrieved 12.4 ± 8.1 vs 10.7 ± 7.5 (p = 0.01), and number of M2 oocytes retrieved 8.1 ± 6.4 vs 6.9 ± 6.0 (p = 0.03), resulting in an increased M2 oocyte yield 63.7% ± 41.3 vs 50.9% ± 30.6 (p = 0.0001). These patients also had a significant increase in clinical pregnancy rate per retrieval 55 vs 41% (p = 0.0016), live birth rate per retrieval 42 vs 34% (p = 0.04) and with no significant difference in multiple gestations or OHSS. A significant improvement in clinical pregnancy rates and live birth per retrieval was detected in patients undergoing curetting. These patients had more embryos frozen, leading to more transfers and more successful pregnancies.

Microbiome affecting reproductive outcome in ARTs

Re: Effects of Advanced Selection Methods on Sperm Quality and ART Outcome: A Systematic Review

Study question Does using a clinical decision support tool, Opt-IVF, to guide hormone dosing during superovulation yield increased clinical pregnancy rates in routine clinical practice? Summary answer Higher clinical pregnancy rates were obtained using Opt-IVF to guide stimulatory hormone dosing during superovulation. What is known already ART success rates and cost depend heavily on the superovulation stage of the cycle. Two prospective single-center trials and one prospective multi-center trial had shown that using Opt-IVF to guide dosing resulted in increased numbers of high-quality embryos, used lower hormone doses, and required less ultrasound testing. This is a retrospective cohort study of the use of Opt-IVF in clinical practice over 12 months compared with the control cohort from the previous 12 months. Study design, size, duration Design: The study examined patients undergoing an IVF cycle using Opt-IVF in a single center for 12 months. Opt-IVF was used in all patients who presented to one of the investigators at Akanksha Hospital during the study period. As a comparison group, we examined patients from the previous 12 months when Opt-IVF was not used, and standard clinical protocols were followed to guide dosing. Duration: The study was conducted over 24 months Participants/materials, setting, methods 204 women aged 25-45 years underwent superovulation using hormone doses suggested by Opt-IVF (intervention). Of the participants, 19% were poor responders, 74% were below 35 years of age, 19% were 35-39 years of age, and 7% were above 39 years of age. The control group included 207 women, of whom 10% were poor responders. 72% were below 35 years of age, 26% were 35-39 years, and 2% were above 39 years of age. Main results and the role of chance The participants in the intervention group required significantly lower cumulative gonadotropin dosage compared to the control group during their controlled ovarian stimulation cycles (p < 0.0001). The average number of total embryos was 20% higher (p < 0.001), and the average number of good-quality embryos was 50% higher (p < 0.0001) in the Opt-IVF group than in the control group. Only 4.9% of cycles were canceled in the intervention group, compared to 12.9% canceled in the control group. Overall, Clinical pregnancy rates (including canceled cycles) increased from 35 % in the control group to 43% in the intervention group. The clinical pregnancy rate for poor responder patients was 39% in the Opt-IVF group but was only 16% in controls (p < 0.0001). Similarly, the clinical pregnancy rates were 34% in patients ages 35-39 years and 33% in patients above 39 years of age, much higher than the corresponding clinical pregnancy rates in controls (27% and 0%, respectively). Higher clinical pregnancy rates were also noted in the intervention group for normal responders and for younger patients. Limitations, reasons for caution This is a single-center retrospective cohort study. Wider implications of the findings Using Opt-IVF to guide dosing resulted in increased numbers of high-quality embryos and higher clinical pregnancy rates, particularly among older patients and poor responders. The proportions of these patients can be expected to grow in the future, so improving results in such patients is essential. Trial registration number not applicable

Study question Does SARS-CoV-2 infection alter human male sex steroid hormones and semen quality? Summary answer SARS-CoV-2 infection reduced circulating testosterone but increased LH and prolactin levels. Also, SARS-CoV-2 infection reduced ejaculate volume, sperm count, concentration, viability, motility, and normal morphology. What is known already SARS-CoV-2 infection has been reported to be associated with a decline in semen quality, although this remains debatable given the novelty of the viral infection. In addition, available data on semen quality and circulating male reproductive hormones in association with SARS-CoV-2 infection is limited and inconsistent. Study design, size, duration This study provides a systematic review and meta-analysis of the impacts of SARS-CoV-2 infection on circulating male sex hormones and semen quality. Participants/materials, setting, methods This study was conducted according to PRISMA guidelines. Following a pre-defined strategic protocol, 852 studies were screened, and 40 articles were identified as eligible for this study. Included studies were from China (7), Germany (1), India (1), Indonesia (1), Iran (6), Iraq (2), Italy (5), Jordan (2), Russia (1), Turkey (12), UK (1), and USA (1). Main results and the role of chance A total of 4807 male subjects were included in the meta-analysis. SARS-CoV-2 infection significantly reduced circulating testosterone and testosterone/LH ratio (an index of Leydig cell function) but increased LH and prolactin levels even after sensitivity analysis when compared to SARS-CoV-2 uninfected patients. Also, SARS-CoV-2 infection significantly reduced ejaculate volume, sperm count, concentration, viability, total and progressive motility when compared to SARS-CoV-2 uninfected patients, although only ejaculate volume and sperm viability remained significant after sensitivity analysis. More so, ejaculate volume, sperm viability, and progressive motility were significantly reduced in SARS-CoV-2-infected patients before treatment when compared with after treatment; nonetheless, only sperm viability, and progressive motility remained significant after sensitivity analysis. Furthermore, ejaculate volume, sperm count, concentration, viability, total and progressive motility were significantly reduced in SARS-CoV-2-infected patients when compared with their pre-COVID state; these alterations, except sperm progressive motility, persisted after sensitivity analysis. Limitations, reasons for caution Although there is an increasing amount of data on the subject matter, SARS-CoV-2-infection is yet novel, and a continuous report and meta-analysis remains pertinent. Also, the included studies are from a few countries, which may not necessarily be a good global representation. Wider implications of the findings This study revealed that SARS-CoV-2-infection significantly reduced circulating testosterone, LH, and semen quality. SARS-CoV-2-induced reduction in semen quality is, at least in part, via a testosterone-dependent mechanism. These findings strengthen existing evidence in the literature on the negative impact of SARS-CoV-2-infection on male reproductive health. Trial registration number N/A

Low semen quality often obligates the use of assisted reproductive technology; however, the association between semen quality and assisted reproductive technology outcomes is uncertain. To further assess the impact of semen quality on assisted reproductive technology outcomes. A retrospective cohort study was carried out at a single academic reproductive medicine center (January 2012-December 2018). Patients undergoing at least one assisted reproductive technology cycle utilizing freshly ejaculated spermatozoa from the male partner were included. We assessed the association between semen quality (as stratified based on WHO 5th edition criteria), paternal age (< or ≥40), and reproductive/perinatal outcomes. To evaluate the differences in assisted reproductive technology outcomes by semen parameters and age, generalized estimating equations were applied for rates of fertilization, pregnancy, implantation, miscarriage, live birth, blast formation, gestational age, and normal embryo biopsy. A total of 2063 couples were identified who underwent 4517 assisted reproductive technology cycles. Average ages of the male and female partners were 39.8 and 37.7, respectively. Lower pregnancy rates were observed in cycles with lower sperm motility (ie <40%; 39.9% vs 44.1%) and total motile count (ie <9 million; 38.3% vs 43.5%). When examining only cycles utilizing Intracytoplasmic Sperm Injection, only a lower motility count was associated with a decline in pregnancy rate (39.1% vs 44.9%). No association was identified between semen quality and gestational age or birth weight. Paternal age was not associated with ART outcomes. However, among assisted reproductive technology cycles in women <40, aneuploidy rate was higher for older men (P<.001). In cycles with women >40, no association between aneuploidy and male age was identified. Sperm motility is associated with pregnancy rates, while other semen parameters are not. In cycles in women <40, paternal age is associated with embryo aneuploidy rate. Paternal factors are associated with assisted reproductive technology outcomes, and future studies should explore mechanisms by which semen quality is associated with assisted reproductive technology outcomes.

Embryo quality and reproductive outcomes of spermatozoa selected by physiologic-ICSI or conventional ICSI in patients with kruger <4% and >4% normo-morphology

Study question Do oxidative parameters in sperm affect oocyte fertilization or blastocyst development in double-donation ICSI cycles? Summary answer While intracellular oxidative stress in sperm does not suppose an impact on fertilization or embryo development, mitochondrial activity is related to increased blastocyst development rates What is known already Reactive oxygen species(ROS) are molecules causing a detrimental effect on sperm quality and function, especially when they are imbalanced with respect to the sperm and seminal plasma antioxidant capacity. Previous research have extensively demonstrated that sperm affected by oxidative stress(OS) show reduced fertilizing capacity, and lead to embryos with impaired development after conventional IVF. The role of OS in sperm in the case of ICSI is, however, controversial because many factors may cause a bias. Not only could this explain the inconsistent results reported thus far, but would also support the need of testing healthy donor gametes to reduce variability. Study design, size, duration This research was devised in the context of a collaboration between a sperm bank and a public university, and was set as a blinded, retrospective study that included samples from 45 sperm donors used in a total of 55 oocyte donation cycles. Intracellular levels of total ROS and of superoxides (O2•-), oxidative stress and mitochondrial activity (MA) were evaluated in frozen-thawed sperm donor samples, and related to the embryo outcomes obtained from the same samples. Participants/materials, setting, methods Samples from selected sperm donors were used to fertilize oocytes through ICSI in oocyte donation programs, and fertilization and blastocyst rates were recorded. Sperm viability and intracellular levels of total ROS and superoxides were determined through a combined assay (2’,7’-Dichlorofluorescin diacetate/Hydroethidine/Sytox Red); oxidative stress and sperm viability were assessed through co-staining with CellRox Far Red and propidium iodide (PI); and MA (mitochondrial membrane potential) and sperm viability were evaluated after staining with DiOC6(3) and PI. Main results and the role of chance The average and standard deviation for fertilization and blastocyst rates were 76.8%±17.3% and 67.5%±22.8%, respectively. Taking the viable sperm population into account, percentages of sperm with high ROS and low O2•- were 17.6%±15.9%, those with high ROS and high O2•- were 0.5%±0.4%, percentages of sperm with low ROS and low O2•- were 25.9%±9.2%, and those with low ROS and high O2•- were 54.5%±21.1%. The evaluation of oxidative stress with CellRox revealed that the percentage of viable sperm with high OS was 63.1%±16.5%. Neither intracellular levels of ROS and superoxides, nor oxidative stress in viable sperm were found to be correlated to fertilization or blastocyst rates (P&amp;gt;0.05). In spite of this, the percentage of viable sperm with high MA, which was 20.7%±10.5%, was observed to be positively correlated to blastocyst rates (Rs=0.276; P=0.04). While sperm mitochondria do not participate in further embryo development, a greater mitochondrial activity in post-thawed donor sperm could indicate a healthier sperm function. Mitochondrial activity could, therefore, be used as a predictive biomarker for embryo development. Limitations, reasons for caution The sample size is the main limitation of this study, despite the efforts made to include a high number of sperm donors. Wider implications of the findings Results obtained in the present study show that ICSI evades the detrimental effects of sperm oxidative stress on fertilization and embryo development. Yet, sperm mitochondrial activity could be used as a biomarker to test the ability of a sperm donor to generate healthy embryos that develop up to blastocyst stage. Trial registration number Not applicable

Study question Does paternal age intensify the negative effects of cryopreservation on sperm quality? Summary answer Paternal age exacerbates the adverse impact of cryopreservation on sperm quality. What is known already Despite mounting evidence indicating age-related declines in sperm quality, the impact of APA on male fertility remains unclear. APA raises questions about DNA fragmentation and mitochondrial activity, a robust indicator of sperm functionality. While sperm cryopreservation is extensively employed in fertility preservation and assisted reproductive technology (ART), its potentially detrimental effects on cells compartments, including mitochondria, persist. The mechanisms of APA on sperm susceptibility to cryopreservation-induced cellular damage still need to be addressed. Study design, size, duration This prospective multicentric study, conducted between January and October 2023, comprised 200 men. Only normozoospermic samples were included in the research (n = 81). Then, based on their age, the participants were categorised into two groups: ages ≤35 (non-APA, n = 40) and ages ≥42 (APA, n = 41). The cohorts were stratified into distinct subgroups, encompassing both fresh and frozen/thawed specimens of APA and non-APA sperm, and subjected to evaluations encompassing mitochondrial functionality, acrosomal reaction, DNA fragmentation and Tyrosine phosphorylation. Participants/materials, setting, methods Semen samples were collected after 2-7 days abstinence and analysed according to WHO 2021 guidelines. Acrosomal status was determined using FITC-conjugate PSA. DNA fragmentation was examined with the Halosperm kit. Mitochondrial functionality was gauged using MitoTracker Red CMXRos through the percentage of stained cells and fluorescence intensity and PCR for mitochondrial DNA copy number (mtDNAcn). Tyrosine phosphorylation was assessed through immunofluorescence and Western Blotting. Main results and the role of chance Non-APA group exhibited significantly superior fresh semen characteristics. DNA fragmentation was notably elevated in fresh semen from APA group, reaching 21.7%, compared to 15.4% non-APA individuals. After cryopreservation, the DNA fragmentation index further increased in men over 42 years to 26.7%. Mitochondrial functionality analysis indicated a significant decrease in staining intensity in cryopreserved sperm, notably in the APA group, with mean fluorescence intensity decreasing from 3.35±1.5 to 1.8±0.5. In contrast, the non-APA group experienced a drop from 3.33±1.6 to 2.53±1.6. Additionally, in the APA group, a significant increase in mtDNAcn was observed in both fresh and frozen/thawed sperm compared to the younger counterparts, indicative of poor mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, with a significant decrease observed in unreacted acrosomes from 71.5% to 57.7% in the non-APA group and from 75% to 63% in the APA group. Furthermore, exploration of capacitation-like changes, like tyrosine phosphorylation, demonstrated a significant decrease with age and a negative impact of cryopreservation on tyrosine phosphorylation in sperm from young males. Limitations, reasons for caution Main limitations are the small number of examined samples and the inclusion of only normozoospermic patients. Therefore, future investigations are needed to provide a more comprehensive understanding of the impact of paternal age on cryopreservation-induced stress in human spermatozoa. Wider implications of the findings Our research sheds light on the influence of paternal age on sperm cryopreservation, revealing higher vulnerability of mitochondria and elevated DNA fragmentation in APA. These findings should be considered when advising older men on choosing cryopreservation techniques in assisted reproductive treatments. Trial registration number Not applicable

Triploid zygote formation rate and ART outcomes

Study question Does the choice of insemination method between conventional IVF (cIVF) and ICSI impact blastocyst euploidy outcomes in relation to cycle characteristics? Summary answer Both cIVF and ICSI yield similar euploidy outcomes regardless of cycle characteristics, suggesting no procedural impact in this cohort. What is known already In non-male factor cases, ICSI has not proven to improve fertilization or live birth rates compared to cIVF, as existing evidence remains conflicting. Moreover, the widespread use of ICSI in non-PGT cycles raises concerns about long-term implications for offspring. Evaluation of euploid rates may help elucidate the relative efficacy of cIVF and ICSI, yet direct sibling oocyte comparisons in PGT scenarios remain particularly limited and relying on small sample sizes (&amp;lt;120 cycles). A large-scale study comparing both methods of insemination in relation to cycle characteristics could provide valuable insights. Study design, size, duration This observational study was performed in a single IVF clinic between October 2017 to December 2024 including 27694 cumulus-oocyte complexes (COCs) across 2161 PGT-A cycles. These COCs were allocated as sibling oocytes to either cIVF or ICSI. The groups are categorized based on the proportion of ICSI inseminated oocytes as follows: 50% (n = 728 cycles), &amp;gt;50-60% (n = 914 cycles), and &amp;gt;60-70% (n = 519 cycles). Participants/materials, setting, methods Couples with female age between 18-49 (mean:36.1 years), and non-severe male factor sperm parameters with a progressive motility ≥65% after capacitation were included. A total of 9132 blastocysts (≥Bl3BCC) were subjected to trophectoderm biopsy on day 5-7 and next generation sequencing (NGS). Cycle characteristics such as female and male age, number of COCs and rate of mature oocytes were analyzed as potential factors associated with fertilization, usable blastocyst formation, and euploidy rates. Main results and the role of chance When comparing outcomes between MIIs following insemination by cIVF (n = 9282) or ICSI (n = 13931) in the different groups, ICSI was not superior to cIVF in terms of fertilization (RR: 1.06 [0.96–1.18], p = 0.235), blastocyst biopsied per inseminated MII (RR: 1.00 [0.80–1.25], p = 0.996) or per 2PN (RR: 0.95 [0.78–1.15], p = 0.581). Similarly, ICSI showed no significant difference compared to cIVF for euploidy per inseminated MII (RR: 1.22, [0.85–1.74], p = 0.285), per 2PN (RR: 1.12 [0.80–1.58], p = 0.499), or per biopsy (RR: 1.09, [0.84–1.43], p = 0.513). Cycle characteristics were independently associated with fertilization, usable blastocyst, and euploidy outcomes, regardless of insemination method. Limitations, reasons for caution The non-random allocation of oocytes between insemination techniques limits the ability to establish causal conclusions. Wider implications of the findings cIVF results in comparable euploidy outcomes to ICSI, making it a viable option for couples in PGT-A cycles, maintaining a balance between efficacy and resource utilization. Trial registration number No

Simple SummaryArtificial insemination (AI) is crucial for dairy cattle worldwide due to its impact on reproductive efficiency and profitability. This study examined the effects of thawing time and temperature, combined with a thermo-resistance test (TRT), on sperm motility and kinematic variables in dairy bulls’ semen for AI. The semen thawing process is critical in maintaining sperm quality, requiring strict adherence to standardized techniques. Incorrect thawing procedures can damage sperm structures. Rapid thawing is necessary to prevent ice recrystallization, and excessive heat exposure can lead to detrimental changes. The thermo-resistance test simulates conditions of the female reproductive tract to assess sperm longevity. Results indicated that Jersey bulls were greater for total and progressive motility percentages compared to Holstein bulls. Jersey bulls exhibited higher values for various velocity parameters and linearity, while Holstein bulls had a lower crossover frequency. The optimal thawing condition was 37 °C for 30 s. Sperm quality decreased with longer post-thawing times. This investigation aimed to analyze the effect that thawing time and temperature in combination with a termo-resistance test had on straws from dairy bulls used for artificial insemination (AI) on semen motility and kinematic variables measured with CASA systems. Eight animals of Holstein and Jersey breeds were used, and nine frozen-thawed semen doses per animal were analyzed for each breed. Three temperatures (35, 37, and 40 °C) and three thawing times (35, 40, and 45 s) were evaluated using a factorial design. Motility and kinematic patterns were analyzed using CASA-mot (Computer-Assisted Semen Analysis of motility) technology at different post-thawing times (0.5, 1, and 2 h). Sperm motility in Jersey bulls was higher (p < 0.05) than in Holstein ones (64.52 ± 1.45% and 53.10 ± 1.40%, respectively). The same effect was seen with progressive motility among the two breeds (Jersey: 45.29 ± 1.00%; Holstein: 36.30 ± 0.98%, p < 0.05). The Jersey breed presented higher values (p < 0.05) of curvilinear velocity (VCL), rectilinear velocity (VSL), average velocity (VAP), linearity on forward progression (LIN), and wobble (WOB). The Holstein breed showed a lower mean value (p < 0.05) of the beat-cross frequency (BCF) compared to the Jersey breed, thus suggesting an effect on VCL and VAP. During the post-thaw period, a gradual increase in VCL was observed at 2 h. VSL and VAP showed a decrease (p < 0.05) as the post-thaw period was prolonged. The study showed differences in sperm quality between Holstein and Jersey breeds, influenced by cryopreservation, thawing, and post-thawing incubation. Thawing at 37 °C for 30 s was considered optimal in relation to sperm motility. In addition, a decrease in sperm quality was observed as post-thawing time increased.

Study question Do in-vitro-matured metaphase I (MI) oocytes have similar developmental potential as in-vivo-matured oocytes in preimplantation genetic testing (PGT) cycles? Summary answer Blastocysts derived from in-vitro-matured MI oocytes demonstrate similar usable blastocyst rates compared to those derived from in-vivo-matured oocytes, offering clinical value in ART cycles. What is known already Approximately 15–20% of oocytes retrieved during ovarian stimulation are immature, often discarded due to their presumed low developmental potential. Rescue in-vitro maturation (IVM) has emerged as a potential method to utilize these immature oocytes, though their clinical utility remains controversial. Prior studies report mixed results regarding the developmental competence of embryos derived from in-vitro-matured MI oocytes. Study design, size, duration This retrospective study included 154 PGT cycles performed between January 2019 and December 2022. A total of 1,810 metaphase II (MII) oocytes were injected, comprising 1,577 in-vivo-matured and 233 in-vitro-matured MI oocytes. Participants/materials, setting, methods PGT cycles included preimplantation genetic testing for aneuploidy (PGT-A), structural rearrangements (PGT-SR), and monogenic disorders (PGT-M). Oocytes retrieved at the MI stage were cultured in vitro for up to 6 hours. Usable blastocysts were defined as euploid embryos suitable for transfer. Embryological and clinical outcomes were compared using statistical models controlling for confounding factors. Main results and the role of chance The usable blastocyst rate was similar between in-vitro-matured MI oocytes and in-vivo-matured oocytes (adjusted RR 0.97, 95% CI 0.40–2.34). However, in-vitro-matured MI oocytes showed lower fertilization (50% vs. 74%, p &amp;lt; 0.001), cleavage (88% vs. 97%, p &amp;lt; 0.001), and blastocyst formation rates (41% vs. 66%, p &amp;lt; 0.001). Importantly, three live births were achieved using embryos derived from in-vitro-matured MI oocytes in patients with no usable in-vivo-matured oocytes. Limitations, reasons for caution This study was retrospective and single-center, with a relatively small cohort of in-vitro-matured oocytes. Further prospective studies are needed to confirm the long-term safety and efficacy of rescue IVM. Wider implications of the findings Rescue IVM offers a valuable opportunity to utilize immature oocytes, potentially increasing the number of viable embryos for patients with diminished ovarian reserve or poor ovarian response. This approach could reduce cycle cancellations and improve clinical outcomes in ART. Trial registration number Yes

Study question Is it possible to score the mosaic embryos in order to prioritise them according to their pregnancy potencial? Summary answer The machine-learning score underscores the crucial role of embryo quality over mosaicism, prioritizing high-quality, day-5 biopsied embryos for enhanced live birth outcomes in ART. What is known already After embryo biopsy and PGT-A analysis, embryos can be classified as euploid, aneuploid, or mosaic, wherein a combination of chromosomally normal and altered cells is detected. The transfer of mosaic embryos has been controversial, but currently, the recommendations of international scientific societies, including ESHRE, advocate for their transfer. In case of pregnancy, a prenatal test is advised to rule out any chromosomal aberration. The non-transfer of these embryos reduces ART success chances, as chromosomally normal children may be born from them. Study design, size, duration The study design is observational and retrospective. A total of 8346 embryos from 2583 PGT-A cycles were considered (January-2017 to January-2023). Only transferred mosaic embryos (n = 263) were included in the study. The trophectoderm biopsies of D5, D6, or D7 blastocysts were analyzed by NGS using the Illumina platform (VeriSeq Illumina®, San Diego, CA, USA). The biopsied embryos were vitrified and transferred in a subsequent cycle. Participants/materials, setting, methods The primary indications for PGT-A included advanced maternal age, abnormal sperm FISH, and recurrent miscarriage or implantation failure. Clinical outcomes, along with variables related to progenitors, embryos, biopsy, ovarian stimulation, and adjuvant treatments, were recorded in a database. Machine learning models were conducted using R statistical software (v.4.3.1), and the ‘Caret’ library was utilized to implement different algorithms. ‘Autoscore,’ as an interpretable scoring tool, combined machine learning and regression modeling. Main results and the role of chance The mean maternal and paternal ages were 34.95±6.38 and 39.13±6.74 years. Biopsies were predominantly performed on day 5 (61%). Additionally, 44% of embryos were of A quality, and 49% were of B quality. Regarding clinical outcomes for mosaic embryos, biochemical, clinical pregnancy, and live birth rates were 51%, 41%, and 36%, respectively. The features used to generate the score included those related to embryo quality, biopsy, and mosaicism. Through 10-fold cross-validation, the study identified top-ranking predictors maximizing the AUC of the predictive model. The highest weight in the score was assigned to embryo quality (42%) and day of biopsy (34%), with lower weights for factors related to mosaicism: monosomy and/or trisomy (18%) and mosaicism level (6%). The machine-learning score indicates that, in transferring embryos with mosaicism, priority should be given to high-quality embryos biopsied on day 5. The type and level of mosaicism have a minor impact on the embryo’s gestational capacity, becoming relevant only when prioritizing embryos of identical quality. In such cases, embryos with lower mosaicism degrees and no monosomy demonstrate higher implantation potential. Limitations, reasons for caution The inherent limitations of a retrospective analysis highlight the need for prospective randomized studies to confirm the validity and usefulness of the mosaic embryo transfer score. Wider implications of the findings A scoring system has been developed to prioritize the transfer of mosaic embryos. The crucial variables are associated with embryo quality and the day of biopsy, reflecting the progression of embryonic development. Conversely, variables related to mosaicism play a secondary role. Trial registration number Not applicable